Glycobiology, or the study of the structure, biosynthesis, and biology of carbohydrates is a significant research frontier. Hexoses, such as galactose, can exist in either the pyranose or furanose form. The understanding of pyranose sugars in biology is emerging, but comparatively less is known about furanose-containing glycoconjugates. The presence of these glycans in various microbes demonstrates their importance. Galactofuranose (Galf) residues are crucial for the virulence or viability of protozoan parasites and fungi. Galf is best known for its role in mycobacteria, where it is an essential component of the cell wall. The mycobacterial cell wall is characterized by an essential arabinogalactan polymer. Recently, the function of a glycosyltransferase, GlfT2, has been established. This enzyme is responsible for polymerizing Galf residues to form the galactan polysaccharide portion of arabinogalactan. Naturally occurring galactan chains consist of alternating [unreadable]-(1?5) and [unreadable]-(1?6) Galf linkages. Interestingly, GlfT2 is responsible for the formation of both of these glycosidic bonds. It is unclear what governs this novel bifunctional activity. This proposal seeks to investigate the molecular basis for this bifunctionality using a combination of chemical and biochemical techniques. Site-directed mutagenesis will be used in conjunction with [unreadable]-(1?5) and [unreadable]-(1?6) linked Galf disaccharide acceptor substrates to ascertain whether GlfT2 uses one or two active sites for glycosidic bond formation. In addition, fluorinated glycosyl acceptor and donor substrates that preclude elongation at either the 5- or 6-position will be synthesized and used to investigate whether the formation of alternating linkages is obligatory for activity of GlfT2. It is anticipated that these studies will provide mechanistic insight into this novel glycosyltransferaseactivity and pave the way towards new antimycobacterial small molecules. PUBLIC HEALTH RELEVANCE: The function of a glycosyltransferase, GlfT2, involved in the formation of galactofuranose polymers in mycobacteria was recently established. GlfT2 catalyzes the formation of two distinct alternating glycosidic bonds. The mechanisms that govern this activity are not understood. This work will investigate the factors that govern this bifunctional activity. It is anticipated that these findings will help guide the development of new antimycobacterial compounds.